Four quantitative PCR (qPCR) assays were evaluated for quantitative detection of
Brachyspira pilosicoli, Lawsonia intracellularis, and E. coli fimbrial types F4 and F18 in pig
feces. Standard curves were based on feces spiked with the respective reference strains.
The detection limits from the spiking experiments were 102 bacteria/g feces for BpiloqPCR
and Laws-qPCR, 103 CFU/g feces for F4-qPCR and F18-qPCR. The PCR efficiency for all
four qPCR assays was between 0.91 and 1.01 with R2 above 0.993. Standard curves, slopes
and elevation, varied between assays and between measurements from pure DNA from
reference strains and feces spiked with the respective strains. The linear ranges found for
spiked fecal samples differed both from the linear ranges from pure culture of the
reference strains and between the qPCR tests. The linear ranges were five log units for F4-
qPCR, and Laws-qPCR, six log units for F18-qPCR and three log units for Bpilo-qPCR in
spiked feces. When measured on pure DNA from the reference strains used in spiking
experiments, the respective log ranges were: seven units for Bpilo-qPCR, Laws-qPCR and
F18-qPCR and six log units for F4-qPCR. This shows the importance of using specific
standard curves, where each pathogen is analysed in the same matrix as sample DNA. The
qPCRs were compared to traditional bacteriological diagnostic methods and found to be
more sensitive than cultivation for E. coli and B. pilosicoli. The qPCR assay for Lawsonia was
also more sensitive than the earlier used method due to improvements in DNA extraction.
In addition, as samples were not analysed for all four pathogen agents by traditional
diagnostic methods, many samples were found positive for agents that were not expected
on the basis of age and case history. The use of quantitative PCR tests for diagnosis of
enteric diseases provides new possibilities for veterinary diagnostics. The parallel
simultaneous analysis for several bacteria in multi-qPCR and the determination of the
quantities of the infectious agents increases the information obtained from the samples
and the chance for obtaining a relevant diagnosis.